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. 2016 Aug 2;11(8):e0158994.
doi: 10.1371/journal.pone.0158994. eCollection 2016.

CFTR Deletion in Mouse Testis Induces VDAC1 Mediated Inflammatory Pathway Critical for Spermatogenesis

Chen Yan  1   2   3 , Qin Lang  1 , Liao Huijuan  1   2   3 , Xie Jiang  1   4 , Yang Ming  1   2   3 , Sun Huaqin  1   2   3 , Xu Wenming  1   2   3
Affiliations

CFTR Deletion in Mouse Testis Induces VDAC1 Mediated Inflammatory Pathway Critical for Spermatogenesis

Chen Yan et al. PLoS One. .

Abstract

Cystic fibrosis is the most common genetic disease among Caucasians and affects tissues including lung, pancreas and reproductive tracts. It has been shown that Endoplasmic Reticulum (ER) stress and heat shock response are two major deregulated functional modules related to CFTR dysfunction. To identify the impact of CFTR deletion during spermatogenesis, we examined the expression of spermiogenesis-related genes in the testis of CFTR mutant mice (CF mice). We confirmed expression changes of MSY2, a germ cell specific RNA binding protein, resulting from deletion of CFTR in testis. Furthermore, real time PCR and Western blot results showed that an inflammatory response was activated in CF mice testis, as reflected by the altered expression of cytokines. We demonstrate for the first time that expression of MSY2 is decreased in CF mice. Our results suggest that CFTR deletion in testis influences inflammatory responses and these features are likely to be due to the unique environment of the seminiferous tubule during the spermatogenesis process. The current study also suggests avenues to understand the pathophysiology of CFTR during spermatogenesis and provides targets for the possible treatment of CFTR-related infertility.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The expression of MSY2, a germ cell specific gene was significantly reduced upon CFTR deletion.
(A) Western blot shows that significantly reduced MSY2 gene expression were detected in Cftr(-/-) mice testis. Right panel is the statistic result. (B) Real time PCR confirmed that MSY2 transcript is also reduced in Cftr(-/-) mice testis compared with wide type mice (** means P<0.01,N = 3). (C) Immunohistochemistry indicate that MSY2 is localized in spermatocyte and spermatid cells. (D) Real time PCR shows that the expression of Msy2 regulated genes, including Acrosin, AKAP4, Cyclin A1 and TB-RBP gene were significantly reduced in Cftr(-/-) mice testis. (* means P<0.05;** means P<0.01,N = 3).
Fig 2
Fig 2. ER and Mitochondrial protein dysfunction may contribute to spermatogenesis defects in CF mice.
(A) The expression of heat shock protein, ER and mitochondrial genes, including Grp78 and VDAC1 were increased in CFTR mutant mice, right panel is the statistic result. (B) Upper panel: Co-IP result confirm that Grp78 could interact with immature band of CFTR in mice testis. Down panel: VDAC1 and Grp78 could interact with each other in mice testis.
Fig 3
Fig 3
(A) ATP measurement shows that Cftr(-/-) mice has significantly increased ATP production compared with WT mice testis. (B) Immunohistochemistry staining of nitrotyrosine, an oxidative stress marker in testis shows that mutant mice has significant increased signal in seminiferous tubule. (C) cytokine profile further confirm that IL-1 level is significantly increased, while TNF-alpha expression shows no significant change in Cftr(-/-) mice testis.
Fig 4
Fig 4. In vitro CFTR knockdown inhibit NF-κB and down-stream targets in NT2 cell model.
(A) Real-time PCR shows that CFTR RNAi could inhibit CFTR transcription successfully, and (B) Knockdown of CFTR increase VDAC1 expression,while P65 expression was inhibited significanltly. (C) Co-IP data shows that Grp78 could interact with VDAC1. The upper two panels are the recipral immunoprecipitation result. The down panel is the Western blot result detecting VDAC1 after immunoprecipitated with VDAC1 antibody. (D) CFTR RNAi could inhibit IL-6 and TNF-alpha expression. (* means P<0.05, ** means P<0.01,N = 3).
Fig 5
Fig 5. Proposed model to explain the specific function of CFTR in germ cell development.
In the current model, CFTR plays a critical role in spermatogenesis through regulation of heat shock proteins and related pathways. We propose that CFTR mutations lead to an over-activated heat shock response, and reduced Grp78 and VDAC1 interactions to initiate the cascade leading to altered energy metabolism and ROS production. Furthermore, over-activated NF-κB activates the pro-inflammatory response and related interleukin increase. Finally, the key RNA binding protein MSY2 expression was significantly reduced, indicating that CFTR affect spermiogenesis through regulation of MSY2 expression.
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The National Basic Research Program of China (2012CB944903).

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